Fully automated diagnostic analyzers are commercially available to perform chemical assays and immunoassays of biological fluids such as urine, blood serum, plasma, cerebrospinal fluid and the like. Generally, reactions between an analyte in a patient sample and reagents used during the assay, result in generating some sort of signal that can be measured by the analyzer. From this signal the concentration of analyte in the patient sample may be calculated.
Heterogeneous immunoassays are popularly used because their versatility allows both large and small sized analytes to be measured. They require three basic steps: (1) binding analyte to a solid phase, (2) separating the unbound sample analyte from the bound solid phase, and (3) measuring the bound analyte. The physical separation step eliminates most interfering substances, thereby providing for a higher sensitivity. Heterogeneous assays include competitive immunoassays and sandwich immunoassays. Diagnostic analyzers generally employ various processing stations, where processing operations such as sample and reagent addition, separate, wash, and mix are performed to accommodate such assays.
In a competitive assay, an antibody to an antigen contained in a first reagent is typically attached to a derivatized magnetic particle, i.e., particles that are responsive to a magnetic field, to make up a solid phase. The second reagent, consisting of antigen attached to a tag and a patient sample are mixed with the solid phase in a test tube. In the absence of patient antigen, some 50% of the antigen-tag is bound to the antibody of the magnetic solid phase. In the presence of patient antigen, some of the antibodies are attached to patient antigen and are unavailable to the tag antigen. As a result increasing amounts of patient antigen leads to decreasing amount of tag antigen. Magnetic separation clusters the magnetic particles of solid phase with the bound tag into a pellet on the side of the tube. The free tag can then be removed by thorough washing and aspiration. Following separation and removal of free tag, another reagent is added so that the amount of bound tag can be measured.
In a typical sandwich immunoassay, multiple steps are used, i.e., an antibody to an antigen is attached to the magnetic particle in high concentration relative to the amount of patient antigen in a sample. Patient antigen is captured by the antibody on the magnetic particles and then the particles (with attached or captured patient antigen) are separated from interfering substances in the sample. A second reagent, containing a second antibody with an attached tag, is added. This second antibody attaches to the patient antigen, captured by the first antibody on the magnetic particle, and results in the formation of a sandwich so that the second antibody tag is held firmly by the antigen to the first antibody on the magnetic particle. At this point, a thorough washing and magnetic separation permit the determination of bound tag which is in proportion to the patient antigen, the excess tag of the second reagent having been removed by the washing action.
In both types of heterogeneous immunoassays, considerable resources and time are required to achieve a sufficiently high degree of washing so as to eliminate interfering constituents and prevent spurious assay results. The degree to which this is achieved in an automated analyzer is an important contributor to the sensitivity of the analyzer.
High throughput is a desirable feature of such analyzers. An important contributor to maintaining a high throughput is the ability to process a plurality of samples through a variety of the different heterogeneous assay process steps that are needed before the signal measurement step may be undertaken. These multiple process steps tend to limit throughput. In the design of new automatic analyzers, in particular those involving complex "sandwich" heterogeneous immunoassays, which often require many separate processing operations, the ability to be capable of detecting a wide variety of analytes yet occupy a minimum of physical space, is an important performance advantage. Generally, repetitive, uninterrupted operation enhances throughput since a number of vessels can be processed simultaneously.
Repetitive operation is particularly difficult to achieve in the case of heterogeneous "sandwich" immunoassays. This is a particularly difficult procedure even when performed manually. It is a particular problem when the automatic wash procedure is incorporated into an automatic analyzer which is capable of performing multiple types of immunoassays. This necessitates the use of multiple wash stations and this is typically accomplished in automatic analyzers by providing such multiple wash stations. For example, the sensitivity requirements of some heterogeneous assays demand that the washing efficiency must be very high. This is normally achieved by repeated washes, each successive wash removes unwanted liquids and reagents so that a progressively lower background level is achieved. At the same time, such a washing regimen places limitations on use of the various processing resources since the analyzer is required to be stationary in a washing activity. Nominally, an assay requires a total of four separate washes interlaced with four separation steps. Accordingly, an important design feature of the analyzer is the ability of non-washing resources to be productively involved with one sample while another is being washed. However, the several mechanisms required to operate individually the several wash stations and the mechanisms required for the wash stations themselves can become quite expensive.
U.S. Pat. No. 4,459,265 assigned to Clinicon describes an automatic analytical apparatus with a stepwise rotatable circular plate. It carries a plurality of reaction tubes on its periphery with several reagent supply stations arranged at different locations around such periphery. The use of multiple stations does provide the machine with the versatility to carry out several different test methodologies, but again does not provide the necessary washing required for heterogeneous immunoassays. The automatic analytical apparatus described in such Clinicon patent, includes a stepwise rotatable circular plate. It carries a plurality of reaction tubes on its periphery with several reagent supply stations arranged at different locations around such periphery. The use of multiple stations does provide the machine with the versatility to carry out several different pre-assay process steps, but again does not provide the necessary washing required for heterogeneous immunoassays.
A conventional chemical analyzer produced by Hitachi, Inc., Tokyo, Japan, Model Number 7050, links together or "gangs" plural wash probes. When this is accomplished, however, the ability of the instrument to perform multiple analytical procedures at the same time is impaired.
U.S. Pat. No. 5,104,808, Laska et al., assigned to the assignee of the present invention, also describes an analyzer which "gangs" wash probes together. The wash probes are ganged together, but also separated into two groups to achieve the goal of providing washed solid support which contains a minimal amount of the original serum/conjugate matrix. According to Laska et al. the wash means includes at least two wash probes coupled or ganged for simultaneous insertion into the reaction vessels at different processing positions. Further, the wash probes are positioned contiguous to the first one of the sample and/or reagent positions in the sequence. The means for adding sample and/or reagents is disabled each cycle for a number of vessels leading and trailing the first and last vessels receiving sample or reagent, the number corresponding to the number of processing positions between the insertable wash probes.
U.S. Pat. No. 5,183,638, Wakatake; discloses an analyzer in which a reaction vessel is conveyed past several devices for adding and agitating magnetic particles as required during a EIA immunoassay.
U.S. Pat. No. 5,192,505, Sakagami, discloses an analyzer in which two different reaction lines are provided, driven by only one driving device, one line adapted to provide for assay operations involved in a calorimetric measurement, the other line adapted to provide for assay operations involved in an immunization agglutination measurement. An advantage of the analyzer is maintenance of a small physical size.
Another feature used in automated analyzers is the scheduling method used to present the sample to the various assay tools. As described in U.S. Pat. No. 5,212,094, an automatic chemical analyzer uses an odd number of reaction vessels disposed in a circular pattern. The reaction vessels are rotated successively half revolutions of the circular pattern plus the distance between reaction tubes. Using such a pattern, it is possible to collectively situate the washing stations proximate one another and thereby facilitate compactness of the analyzer, however such an analyzer is not capable of performing heterogeneous immunoassays.
The analyzer of U.S. Pat. No. 5,352,612 includes a movable sample support for holding samples arranged in a first, plurality of positions for movement in a first direction. An indexing drive for the sample support moves the samples in the sample support in the first direction in a set of increments wherein each increment represents a movement of the samples an amount corresponding to a number of samples. Such a system enables separation of logical space from physical space in the system, allowing more freedom in placement of mechanical equipment while permitting proper sequencing of operations both in space and in time. This system requires a very complex time-indexing motion of a rotatable sample support that is determined differently for each and every loading of the sample support. As best understood, the movement pattern is determined by the number of pairs of reaction cuvettes. The pattern may vary depending on the assay to be performed and the times spent stopped before different processing resources varies according to the operation of the processing resource.
U.S. Pat. No. 5,380,487, assigned to Pasteur Sanofi Diagnostics, describes an analyzer in which assay resources are assigned fixed operating sequence which begin and end within a time cycle of fixed duration. When different samples having different assay protocols are entered into the analyzer, assay resource requirements for the different samples are determined and "time slots" of the required resources are allocated thereto. Provision is made for handling heterogeneous assays, however because an incubator belt physically intersects the wash wheel, a complex relationship is established between the indexing time of an incubator belt, the indexing time of a wash wheel, and the cycle time of operations performed on reaction vessels as they are moved along the wash wheel. In addition, the control means must determine which time-based assay resources are required to process a test and then check the availability of those assay resources on a cycle-by-cycle basis against the allocation of the resources to the processing of other tests underway. Absent any conflicts in the allocation of assay resources, processing of a reaction vessel will sequentially follow a preceding reaction vessel. Consequently, initiation of the sample processing for a test may be delayed until all the necessary assay resources are available for processing. Although such scheduling may reduce the overall number of indexing cycles necessary to complete the processing of all the tests, scheduling conflicts do delay processing initiation and also result in processing resources being idled.
From a study of the different approaches taken in the prior art to the problems encountered with automated processing of complex heterogeneous immunoassays, there is a need for improved automated analyzers and associated processes for handling samples. At the same time, there is a need for maintaining a high throughput without introducing complex programming and, at the same time, minimizing the physical size of an analyzer. In particular, there is a need for a method of providing bound-unbound analyte separation and wash in a minimum of processing time, in a pattern that enhances/maintains throughput of an automated analyzer.
It is an object, therefore, of the present invention to provide an apparatus for providing the operations required in heterogeneous immunoassays at an increased efficiency and with greatly simplified sample handling techniques.